Biomedical subjects

M L Meistrich

Publications and source records attributed to M L Meistrich.

At least 37 records · Page 2Linked to original sources

PubMed1999

Hormonal treatment after cytotoxic therapy stimulates recovery of spermatogenesis.

Previous studies have shown that treatment of rats with gonadotropin-releasing hormone (GnRH) analogues or steroids either before exposure to procarbazine or radiation or after irradiation enhances subsequent levels of spermatogenesis. We demonstrate here that giving a GnRH agonist after procarbazine injection also enhances spermatogenesis and fertility. We also demonstrate that GnRH agonist stimulated recovery of spermatogenesis and fertility not only when the hormone was administered immediately after irradiation, but also at 20 weeks after irradiation, after the decline in spermatogenesis had occurred. These results suggest that GnRH agonist treatment given to azoospermic men after cytotoxic therapy for cancer may stimulate the recovery of spermatogenesis and fertility.

Animals↗

PubMed1999

Restoration of spermatogenesis by hormone treatment after cytotoxic therapy.

Cytotoxic therapy is effective in treating various cancers, but many of the agents used can cause sterility in men, regardless of the age at which they were treated. Originally, it was thought that infertility in individuals who had undergone cytotoxic therapy resulted from the destruction of stem spermatogonia. However, studies using rats suggest that some stem spermatogonia can survive cytotoxic therapy and that infertility is caused by the inability of these spermatogonia to differentiate. The author believes that the failure of these cells to differentiate may be related to the high levels of intratesticular testosterone encountered in these situations. In rats that have previously received cytotoxic therapy, the administration of a gonadotrophin-releasing hormone (GnRH) agonist restores the ability of spermatogonia to differentiate, and as a result normal spermatogenesis returns. This finding suggests that GnRH agonists show promise as a treatment for male sterility induced by cytotoxic therapy, although further studies are required.

Animals↗

PubMed1999

Human Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of cyclic AMP-induced transcription for proteolysis.

Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIgamma, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIgamma and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIgamma- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B-/-) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1-Cullin-F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.

Activating Transcription Factors↗

PubMed1999

Stimulation of spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mutant mice by gonadotropin-releasing hormone antagonist treatment.

Male juvenile spermatogonial depletion (jsd) mutant mice are sterile because of spermatogenic failure and so may provide a model for genetically caused human male infertility. To test the effects of testosterone suppression therapy on spermatogenesis in jsd/jsd mice, we treated them with Nal-Glu, a GnRH antagonist. Treatment with Nal-Glu at 2500 microg/kg/day was started at 5.5 or 8 weeks of age and continued for 4 or 8 weeks. Differentiation of spermatogonia was evaluated by the percentage of tubules containing two or more spermatocytes (% of differentiating tubules). Nal-Glu treatment caused a marked decrease in the weights of the testes and seminal vesicles and intratesticular testosterone concentrations. However, in contrast to a value of 1.3% in untreated jsd/jsd mice, the mean % of differentiating tubules was 59.9% and 25.1% in treatment groups started at 5.5 and 8 weeks of age, respectively. We propose that spermatogonial differentiation in jsd/jsd mutant mice is sensitive to the high intratesticular levels of testosterone and can only proceed when testosterone production is suppressed.

Animals↗

PubMed1999

The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis.

Sertoli cells, the supportive cells in the seminiferous epithelium, orchestrate spermatogenesis by providing structural and nutritional support to germ cells. In the rat, physiological apoptosis occurs continuously to limit the size of the germ cell population to numbers that can be adequately supported. This form of germ cell death is exaggerated after testicular insults such as toxicant treatment, radiation, and heat exposure. The Fas system has been proposed as a key regulator of the activation of germ cell apoptosis. According to this model, Fas ligand (FasL) and Fas, expressed by Sertoli cells and germ cells, respectively, respond to environmental conditions and initiate germ cell death. To assess the role of the Fas system in various testicular injury models, a semiquantitative RT-PCR technique was used to evaluate the expression kinetics of both FasL and Fas after induction of massive germ cell death. Radiation exposure, which targets actively dividing germ cells, produced an up-regulation of Fas gene expression, but not FasL gene expression. However, administration of mono-(2-ethylhexyl)phthalate and 2,5-hexanedione, two widely studied Sertoli cell toxicants, resulted in up-regulated expression of both FasL and Fas. These data support the following hypotheses: 1) up-regulation of Fas is a common and critical step for initiating germ cell death in vivo; and 2) if Sertoli cells are injured, Sertoli cells up-regulate FasL to eliminate Fas-positive germ cells, which cannot be supported adequately.

Animals↗

PubMed1999

Hormone pretreatment enhances recovery of spermatogenesis in rats after neutron irradiation.

Previous studies showed that a 6-week pretreatment of rats with testosterone plus estradiol enhanced the recovery of spermatogenesis 9 weeks after gamma irradiation, resulting in a dose-modifying factor (DMF) of about 2. To test whether the effect of the hormone treatment was mediated through changes in oxygen tension, thiol levels or DNA repair, we irradiated the testes of rats with neutrons, which depend less on these factors than does low-LET radiation for their cytotoxic action. Control rats and rats treated with testosterone plus estradiol were irradiated with 0.7-2.7 Gy of cyclotron-generated high-energy neutrons. The recovery of spermatogenesis was assessed 9 weeks after irradiation by testis weights, sperm counts and the tubule repopulation indices. Greater recovery of spermatogenesis was observed for all end points, with a DMF of about 2 for rats treated with testosterone plus estradiol compared to the irradiated, cholesterol-treated rats. The equal protection factors for neutrons and gamma rays indicate that oxygen, thiols and repair of DNA damage are unlikely to be involved in the protective effect of the hormone treatment.

Animals↗

PubMed1998

Ubiquitination of histone H3 in elongating spermatids of rat testes.

Because of the potential role of histone ubiquitination in altering chromatin structure, we characterized the levels of ubiquitination of specific histones in meiotic and postmeiotic germ cells in rat testes by two-dimensional gel electrophoresis. The levels of the major ubiquitinated histone forms, mono- and poly-ubiquitinated H2A, were highest in the pachytene spermatocyte stage, declined thereafter through the round spermatid stage, and reached their lowest levels in elongating spermatids. Three additional ubiquitinated histone species, besides H2A, were detected using anti-ubiquitin antibodies specifically in the fraction enriched in elongating spermatids. Based on their electrophoretic mobilities, they corresponded to uH3, uTH3, and uH2B. Polyubiquitinated forms of these proteins were also observed. The identity of these proteins was confirmed by immunoblotting with anti-H3 antisera and by differential extraction of the proteins from the nucleus with increasing salt concentrations. This is the first report of ubiquitination of H3 in vivo. We speculate that its ubiquitination could loosen the nucleosome structure in preparation for histone removal, be a consequence of nucleosome relaxation or disruption caused by other means, or target H3 for degradation.

Animals↗

PubMed1998

Mouse protamine genes are candidate targets for the novel orphan nuclear receptor, germ cell nuclear factor.

Proper expression of the protamine genes is an important event in the terminal differentiation of the male gametes in mammals. Here we present evidence that the novel orphan member of the nuclear receptor gene superfamily, Germ Cell Nuclear Factor (GCNF), may play a role in the regulation of these genes. Previously, we have shown that high-level expression of GCNF mRNA is restricted to spermatids (stages 1-8) in the adult male mouse, which makes it temporally and spatially available to regulate the mouse protamine genes. Furthermore, we have previously identified a sequence to which GCNF can bind, which consists of a direct repeat of the core halfsite AGGTCA with zero base pairs spacing the repeats (DRO). We have identified several genes that contain DRO sequences in their 5' promoter regions, including the protamines. The mouse protamine 1 and 2 (Prm1 and Prm2) genes therefore are potential target genes for GCNF regulation. We show that GCNF binds to one of the two DRO sequences in the Prm1 promoter, and to the DRO sequence in the Prm2 promoter in a specific manner. Furthermore, by using antibodies directed against GCNF, we detect endogenous GCNF in testis nuclear extracts and elutriated round spermatid nuclear extracts in Western blots. Additionally, by using these antibodies in gel-shift assays, we show that this endogenous GCNF can bind to both the Prm1 and Prm2 promoters. This evidence supports the hypothesis that GCNF mediates a novel signaling pathway, two targets of which may be the Prm1 and Prm2 genes in spermatids.

Animals↗

PubMed1998

Hormonal stimulation of the recovery of spermatogenesis following chemo- or radiotherapy. Review article.

Radiation and chemotherapeutic drugs produce prolonged depression of sperm counts in rodents and humans. Previously, three approaches have been developed in experimental animals that have had some success in preventing or reversing this toxicity. These approaches included pretreatment with hormones that suppress spermatogenesis, stimulation of stem cell number, and supplementation with testosterone. A different rationale for the ability of particular hormonal treatments to reverse prolonged azoospermia is presented in this review. In many cases prolonged azoospermia occurs even though the stem spermatogonia survive the toxic insult, but the differentiation of these spermatogonia to produce sperm fails. In the rat, the block appears to be at the differentiation of the A spermatogonia. Hormone treatments with testosterone or with GnRH agonists, which suppress intratesticular testosterone levels, relieve this block and result in the production of differentiating cells. When the hormone treatment is stopped the production of differentiating cells continues, mature sperm are produced, and fertility is restored. If a similar mechanism can be demonstrated to hold in humans, the fertility of men who have been rendered infertile by treatments for testicular and other cancers could be improved.

Animals↗

PubMed1998

Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in p53-deficient mice.

The effect of the p53 gene on the survival of mouse testicular cells was evaluated by analysis of degenerating and terminal transferase-mediated end labeling (TUNEL)-positive cells and the subsequent production of further differentiated progeny. In p53 null mice, in contrast to wild-type mice, radiation induced negligible levels of degenerating or TUNEL-positive differentiating spermatogonia within 24 h. This was correlated with higher production of differentiated progeny of the differentiating spermatogonia in p53 null mice. Contrary to the differentiating spermatogonia, the stem spermatogonia of p53 null mice produced fewer differentiated progeny after irradiation than did the stem cells of wild-type mice. We conclude that, because the degeneration and TUNEL positivity of the differentiating spermatogonia in mice of different genotypes were correlated with each other and were dependent on p53, this process is indeed apoptosis. In the differentiating spermatogonia, p53-dependent apoptosis accounted for the bulk of the loss of their progeny after irradiation. Furthermore, whereas the differentiating spermatogonia died by apoptosis that was dependent on p53, the stem spermatogonia, which are more radioresistant, did not.

Animals↗

PubMed1997

Supraadditive apoptotic response of R3327-G rat prostate tumors to androgen ablation and radiation.

PURPOSE: Androgen ablation is often combined with radiation in the treatment of patients with prostate cancer, yet, the optimal sequencing and the mechanisms governing the interaction are not understood. The objectives were to determine if cell killing via apoptosis is enhanced when the combined treatment is administered and to define the relationship of changes in this form of cell killing to tumor volume growth delay. MATERIALS AND METHODS: Dunning R3327-G rat prostate tumors, grown in the flanks of Copenhagen rats, were used at a volume of approximately 1 cc. Androgen ablation was initiated by castration, and androgen restoration was achieved with 0.5 cm silastic tube implants containing testosterone. 60Co was used for irradiation. The terminal deoxynucleotidyl transferase (TUNEL) histochemical assay was used to quantify apoptosis. RESULTS: Tumors from intact and castrate unirradiated control rats had average apoptotic indices (percent of apoptotic cells) of 0.4 and 1.0%, respectively. The apoptotic index varied only slightly over time (3 h to 28 days) after castration (range 0.75-1.43%). Irradiation of intact rats to 7 Gy resulted in a peak apoptotic response at 6 h of 2.3%. A supraadditive apoptotic response was seen when castration was initiated 3 days prior to 7 Gy radiation, with peak levels of about 10.1%. When the radiation was administered at increasing times beyond 3 days after castration, the apoptotic response gradually diminished and was back to levels seen in intact rats by 28 days after castration. Tumor volume growth delay studies were consistent with, but not conclusive proof of, a supraadditive effect when the combination was used. DISCUSSION: A supraadditive apoptotic response was seen when androgen ablation and radiation were used to treat androgen sensitive R3327-G rat prostate tumors. This supraadditive effect was dependent on the timing of the two treatments. Further studies are required to more fully define the optimal timing and administration of androgen ablation and radiation.

Animals↗

PubMed1997

Quiescence in R3327-G rat prostate tumors after androgen ablation.

Androgen ablation is frequently used in conjunction with radiotherapy in the treatment of high-risk prostate cancer. Androgen ablation-induced cell kinetic changes could result in sub-additive (increased quiescence) or supra-additive (reduction in repopulation) interactions with radiotherapy. The cell kinetic changes were studied in R3327-G Dunning rat prostate tumors grown in vivo using double thymidine analogue labeling and flow cytometry, the terminal deoxynucleotidyl transferase-mediated nick end labeling assay for apoptosis, and measurements of tumor cell numbers. Tumors grown in intact and castrate male rats were continuously labeled for various periods of time with chlorodeoxyuridine and pulse-labeled with iododeoxyuridine 8 h before tumor removal. Androgen ablation resulted in a maximal reduction in labeling index (10 to 1.6%) and an increase in potential doubling time (Tpot; 6-42 days) within 3 days, which was related to a reduction in growth fraction (65% to <10%). In contrast, the length of S-phase was minimally altered (19 to 23 h). The response to androgen ablation involved little apoptosis and no necrosis, and Tpot was approximately the same as the tumor volume doubling time. Hence, the increase in Tpot was mainly the result of a shift to quiescence, and this shift occurred with minimal cell loss. Because quiescence is usually associated with radioresistance, these cell kinetic changes suggest that a sub-additive interaction may occur for some prostate cancers when androgen ablation and irradiation are given together.

Animals↗

PubMed1997

Protection from procarbazine-induced testicular damage by hormonal pretreatment does not involve arrest of spermatogonial proliferation.

Hormone treatments that suppress sperm production enhance the recovery of spermatogenesis after gonadal exposure to various cytotoxic agents. It has generally been assumed that the mechanism of protection involved an arrest of spermatogonial kinetics. To test this hypothesis critically, we examined spermatogonial kinetics and numbers in rats in which the completion of spermatogenesis was suppressed with a 6-week testosterone plus 17beta-estradiol treatment that protected the testis from procarbazine-induced damage. Histological examination showed that the numbers of A-aligned, intermediate, and B spermatogonia and preleptotene spermatocytes and their mitoses were unaffected by testosterone plus 17beta-estradiol treatment. Flow cytometric analysis of bromodeoxyuridine-labeled cells showed that the percentage of diploid cells undergoing DNA synthesis, the progression of B spermatogonia and preleptotene spermatocytes through S-phase, the division of intermediate and B spermatogonia, the entry of intermediate spermatogonia into their next S-phase as type B cells, and the progression of cells through meiotic prophase were either unchanged or very slightly increased. Thus, changes in spermatogonial numbers or suppression of their proliferation cannot account for protection of spermatogenesis from exposure to cytotoxic agents.

Animals↗

PubMed1997

Chemotherapy induces transient sex chromosomal and autosomal aneuploidy in human sperm.

Each year more than 20,000 children and young persons of reproductive age are exposed to known mutagens in the form of chemo- and/or radiotherapy for cancer in the States. As more of these treatments are effective there is growing concern that genetic defects are introduced in the germ cells of these young patients. It is well documented for male rodents that treatment with chemo- and radio-therapeutic agents before mating can cause genetic damage in the germ line, and the magnitude of heritable effects depends on the spermatogenic cell stage treated. Similar germinal effects are suspected to occur in humans but remain unproven. Hodgkin's disease (HD) is an example of a malignancy which is typically diagnosed during a patient's reproductive years. In our study we observed eight male HD patients who were treated with NOVP (Novanthrone, Oncovin, Vinblastine, Prednisone) chemotherapy. We evaluated sperm aneuploidy using multi-colour fluorescence in situ hybridization (FISH), and found approximately 5-fold increases in sperm with disomies, diploidies and complex genotypes involving chromosome X, Y and 8. Increases in sex chromosome aneuploidies arose from segregation errors at meiosis I as well as meiosis II. The aneuploidy effects were transient, however, declining to pretreatment levels within approximately 100 days after the end of the therapy. When compared with normal men, some HD patients showed higher proportions of certain sperm aneuploidy types even before their first therapy.

Adult↗

PubMed1997

Rapid recovery of spermatogenesis after mitoxantrone, vincristine, vinblastine, and prednisone chemotherapy for Hodgkin's disease.

PURPOSE: Because the effects of mitoxantrone on human male fertility were unknown, we determined prospectively the effects of three courses of mitoxantrone (Novantrone), vincristine (Oncovin), vinblastine, prednisone (NOVP) chemotherapy on the potential for fertility of men with Hodgkin's disease (HD). PATIENTS AND METHODS: Semen analyses were performed on 58 patients with stages I-III HD before, during, and after chemotherapy and after the sperm count recovered from the effects of abdominal radiotherapy that was given after chemotherapy. RESULTS: Before the initiation of treatment, 84% of the patients were normospermic. Sperm counts declined significantly within 1 month after the start of NOVP chemotherapy. In the month after chemotherapy, 38% of patients were azoospermic, 52% had counts < 1 million/ mL, and 10% had counts between 1 and 3 million/mL. Between 2.6 and 4.5 months after the completion of chemotherapy, sperm counts recovered rapidly to normospermic levels in 63% of patients. In the remaining patients who were followed up for at least 1 year after standard upper abdominal radiotherapy, counts also recovered to normospermic levels. CONCLUSION: NOVP chemotherapy, like most other regimens, produced marked temporary effects or spermatogenesis. However, sperm production recovered very rapidly, within 3 to 4 months after the end of NOVP chemotherapy. This pattern was caused by killing differentiating spermatogenic cells, but there was little cytotoxicity or inhibition of stem cells from mitoxantrone or the other drugs. After the combination of NOVP plus abdominal radiotherapy, sperm counts and motility were restored in most patients to pretreatment levels, which were compatible with normal fertility.

Adult↗

PubMed1997

During development, 17alpha-estradiol is a potent estrogen and carcinogen.

Neonatal administration of estradiol-17beta (E2-17beta) increases the nuclear DNA content in the mouse reproductive tract. Similar responses have been demonstrated for synthetic estrogens such as diethylstilbestrol. One of the questions raised regarding environmental estrogens such as organochlorines is whether they are potent enough to result in abnormal changes such as those demonstrated by both natural and synthetic estrogens. To test this hypothesis, female BALB/c mice were treated neonatally (days 1-5) with either E2-17beta or estradiol-17alpha (E2-17alpha), an inactive stereoisomer in adult reproductive tissues. We also proposed whether neonatal administration of (E2-17alpha) was tumorigenic and whether the effects were age dependent. To answer these questions, one set each of 10 day-old treated and control mice received short-term secondary administration of E2-17beta, E2-17alpha, or cholesterol. Cervicovaginal tracts from intact BALB/c mice were examined histologically and by flow cytometry at 70 days of age and by histology alone at 18 to 22 months of age. The results include several important findings: a) like E2-17beta, neonatal E2-17alpha treatment induced persistent vaginal cornification, hypospadias, vaginal concretions, and hyperproliferation in nearly 100% of the animals regardless of the secondary treatment for most groups; b) neonatal E2-17alpha treatment increased the nuclear DNA content of cervicovaginal epithelium at one-half both the level (mean DNA index of 1.02 vs 1.04) and incidence (22 vs 46% of the animals) of E2-17beta; c) short-term secondary treatment with E2-17alpha, unlike E2-17beta, did not significantly augment the increase in DNA content (13% for E2-17alpha vs 37 and 56% for control and E2-17beta, respectively); and d) neonatal administration with E2-17alpha induced adenosquamous tumors in the reproductive tract in 25% of the animals. Therefore, the biological effects (estrogenic potency) of E2-17alpha may be age dependent.

Animals↗

PubMed1997

Radiation-induced cell death in the mouse testis: relationship to apoptosis.

The killing of male germ cells by radiation and other toxicants has recently been attributed to apoptosis, but a critical evaluation of the presence of the different features of apoptosis has not been performed. In this study, mouse testes exposed to radiation were examined by light microscopy, electron microscopy and terminal transferase-mediated end labeling (TUNEL) to determine whether the cells were apoptotic according to several criteria. Testes were irradiated with single doses of gamma rays of up to 5 Gy. Although the maximum response was produced by 5 Gy, even 0.5 Gy induced marked changes. The numbers of abnormal spermatogonia reached a peak 12 h after irradiation and then declined, and the total number of spermatogonia began to decline at 12 h. These changes were most prominent among the B spermatogonia and early preleptotene spermatocytes. When examined by both light and electron microscopy, the majority of the abnormal spermatogonia showed condensation of nuclear chromatin and some showed features similar to necrosis, but the typical morphological characteristics of apoptosis, margination of chromatin and nuclear fragmentation, were rare. Many of the abnormal spermatogonia were TUNEL-positive, with the maximum number occurring at 12 h after irradiation. Although the morphological features of radiation-induced spermatogonial degeneration were not typical of apoptosis, the TUNEL staining, the rapid onset of degeneration and the sensitivity to low doses suggest that the mechanism of radiation-induced spermatogonial degeneration is closely related to apoptosis.

Animals↗

PubMed1996

Failure of spermatogenesis to recover despite the presence of a spermatogonia in the irradiated LBNF1 rat.

The dose and time response of LBNF1, rat testis to gamma irradiation was studied with use of single doses from 2.5 Gy to 6.0 Gy. Germ cells were initially depleted as a result of killing the radiosensitive differentiating spermatogonia. Some recovery of spermatogenesis was observed at 4 and 6 wk after irradiation as indicated by the repopulation of tubules with germ cells derived from surviving stem spermatogonia. Although spermatogenesis showed additional recovery and was maintained throughout the 60-wk follow-up period after 2.5 Gy, at doses from 3.5 Gy to 6.0 Gy, repopulation indices declined after 6 wk to less than 2%. The numbers of Sertoli cells per nonrepopulating tubule were constant, independent of radiation dose or time. In addition, the nonrepopulating tubules contained an average of one A spermatogonium per 100 Sertoli cells. The size and shape of these cells corresponded to undifferentiated A spermatogonia in nonirradiated control tests. Despite high labeling (40%) and mitotic (20%) indices, the numbers of A spermatogonia changed very little with time, and no differentiated cells were produced in these tubules. The failure of spermatogenesis to recover was not due to hormone deficiency: serum gonadotropin levels increased after irradiation, and serum testosterone remained at control levels. The irradiated LBNF1 rat model may be useful for studying the regulation of differentiation of A spermatogonia.

Animals↗